The long-term objective of this research is to elucidate the molecular mechanism for the regulation of calcium signaling that follows the stimulation of phosphoinositide-specific phospholipase C. This signaling pathway serves important roles in regulating cellular activities in response to extracellular stimuli and thus is essential for the understanding of cell function under normal and pathological conditions. Capacitative Ca2+ entry is an inevitable step of the phospholipase C-mediated pathway. However, it remains to be established what molecules form the store-operated channels and how they become activated. Recent studies showed that mammalian homologues of Drosophila Trp (transient receptor potential) formed store-operated channels and human Trp3 was activated via conformational coupling by the activated inositol 1, 4, 5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). The Trp protein was also physically associated with IP3Rs and with RyRs in vivo. Two Trp-binding domains and one IP3R-binding domain have been identified from the N-terminus of an IP3R and the C-terminus of Trp3, respectively. Expression of these domains in HEK 293T cells interferes store- operated Ca2+ entry. In addition, the IP3R-binding domain of Trp3 also binds calmodulin in a Ca2+-dependent manner and the interaction with IP3R and that with calmodulin are mutually exclusive. In excised inside-out patches from cells expressing Trp3, a peptide representing the Trp binding domain of IP3R activated Trp3 to the same extent as removing or inactivating calmodulin. The channel was inactivated by the reassociation of calmodulin. Thus, displacing calmodulin may be an essential step for conformational coupling of Trp channels by IP3Rs and RyRs. The aims of the proposed research are: 1) to identify additional binding sites for conformational coupling from IP3Rs, RyRs, and Trps, 2) to investigate conditions that affect the binding affinities, and 3) to study the function of each binding domain. We will use GST pull-down assay and the yeast two-hybrid system to study the binding and use electrophysiological methods and Ca2+ imaging techniques to study the functions of the binding domains. Results from these studies will greatly enhance our understanding of the molecular mechanism of intracellular Ca2+ regulation by the influx through Trp and native store-operated channels.